Hand, foot and mouth disease (HFMD) is one of the common infectious diseases caused by enterovirus. The highest morbidity is found in infants under 5 years old. In every year, June to August is the epidemic season, and the main routes of transmission are through the respiratory tract and oral infection. HFMD has characteristics of strong infection, wide route of transmission, rapid transmission, big epidemic intensity, and it can cause pandemics in a short period of time. Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the main pathogens of HFMD. As compared with CA16, EV71 is much more dangerous, and more than 90% of severe cases and death cases are caused by EV71 virus. EV71 can invade the vital organs such as heart, brain, and kidney, resulting in serious complications, such as acute flaccid paralysis, encephalitis, aseptic meningitis, myocarditis, hepatitis, neonatal septicemia, and the like, which pose a serious threat to the physical and mental health of children. In recent years, the incidence of hand, foot and mouth disease persists to ascending in China. A data published by the Ministry of Health shows that a total of more than 2,198,000 incident cases of HFMD with 569 deaths are reported in China in 2012, and there is an increase in both the morbidity and mortality as compared with the data in 2011. Viewed from recent years' situation, the number of patient in China increases each year, grows fast, has a wide distribution, and most of the patients are in undeveloped areas, which bring huge loss to the country and also affect the social security. HFMD has become a focus of the party and the government as well as the people of concern.
At present, there are neither vaccines in the market useful for preventing HFMD, nor drugs effective in treating infected patients in the world. Although there are international reports on the research progress of subunit vaccines, DNA vaccines and attenuated live vaccines, none of them can achieve a satisfactory effect on animal protection. Some attenuated live vaccines have risk of reverting to virulence and they can also cause mild neurologic clinical symptoms, and thus the safety of a vaccine remains uncertain. It is currently known that all EV71 vaccines declared to the State Food and Drug Administration are all inactivated vaccines, and such vaccines have some disadvantages such as certain destroyed antigen targeted, poor immunogenicity and excessive dosage, and the doubtful applicability of currently used animal model. Experiments in vitro show that the inactivated EV71 whole virus vaccine has no neutralizing effect on many EV71 strains isolated from the clinical samples; further, it has no protection effect in the infected immature rat model; and the inactivated vaccine has risk of being inactivated incompletely. Therefore, development of a prophylactic EV71 vaccine with high cost-effectiveness is of important significance for preventing HFMD infected with EV71.
The emergence of Virus-like Particle (VLP) vaccine provides a new opportunity to develop a novel, safe and efficient vaccine. VLP vaccine expresses one or more structural proteins of a virus by molecular biotechnology. These structural proteins have an ability of native self assembly, and they can form a steric configuration and an antigenic epitope similar to those of the naturally occurring virus particle, but they do not carry a viral nucleic acid, have strong immunogenicity, and have no infectivity, and thus have no risk of inactivated incompleteness or reverting to virulence. Meanwhile, there are virus antigens with high density on the surface of vaccines which retain the conformational epitopes, and the VLP vaccine can be delivered to immune cells through the same route as the whole virus vaccine to effectively induce the body immune system, in order to produce a protective immune response. In this regard, the VLP vaccine is also superior to the inactivated vaccine in which some important antigenic determinants may be destroyed during inactivation. In addition, another advantage of the VLP vaccine is that it can be engineered arbitrarily as required so as to make it better stimulate the body immune system to produce a protective immune response.
EV71 are small RNA virus, viral genome of EV71 is a single stranded RNA of about 7.4 kb. The virus particle is an icosahedron sphere structure that is three-dimensionally symmetric, without envelope and protrusion, and has a diameter of about 24-30 nm. The capsid of the virus particle consists of 60 subunits, and each subunit has a pentamer-like structure which is assembled by 4 capsid proteins (VP1-VP4). Studies show that four structural proteins of EV71 can self assemble into a structure of VLP in cells, which has a spatial structure similar to the naturally occurring virus. Currently, many research institutions at home and broad are all developing EV71 virus-like particle (EV71 VLP) vaccines. The assembly of EV71 is carried out by co-expression of viral protease 3CD and P1 precursor protein, the process of P1 precursor protein by 3CD protease to produce 4 structural proteins, and the self assembly of 4 structural proteins into a VLP, which does not carry viral nucleic acid, has no potential risk of getting cancer, and has good safety, immunological property and biological activity, furthermore, it can be prepared and purified in a large scale. Thus, VLP vaccines have been a main direction for developing prophylactic EV71 vaccines.
A key to develop EV71 VLP prophylactic vaccines is that VLP samples can be prepared efficiently in a large scale, and the prokaryotic expression system and the eukaryotic expression system are currently the most commonly used expression system.
Most proteins expressed in the prokaryotic expression system lose their natural conformation, and thus they cannot produce protective antibodies. On the other hand, most expression products may be inclusion bodies. The inclusion bodies have complex denaturation and renaturation steps, big loss of proteins, and low yield, such that it is difficult to achieve the large scale production. There are also examples of soluble expression, but the expression level is low and it is difficult to purify the proteins of interest. It is also reported that fusion and expression are performed to increase the solubility and expression level of the interest proteins, although it is relatively easy to perform purification, the cleavage of fusion proteins needs expensive enzymes, such that large scale production cannot be achieved.
The commonly used eukaryotic expression systems include mammalian cell expression system, insect baculovirus expression system and yeast expression systems. Proteins can spontaneously form VLP in the eukaryotic expression system, providing a great convenience for the purification process. However, since VLP is prepared mainly using insect cells, it needs a high cultural condition and has complicated purification process, thereby limiting the need for large scale production; furthermore, baculovirus-insect cell expression system produces baculovirus particles and other pollutants that significantly influence the effect of vaccine, it is very difficult to separate baculoviruses from the prepared VLP, and the measures such as inactivated treatment are required, which have great effect on the quality of vaccines. The mammalian cell expression system plays a role in the process, modification and correct fold of the protein after expression, but it is difficult to artificially control the mammalian cell expression system and the cost for the control is high, and thus the application of the mammalian cell expression system is limited. Hansenula polymorpha expression system not only has characteristics of stable heredity, simple operation, being easy to high density culture, high production levels of foreign proteins, low production cost, being suitable for industrial production, and the like, but also has advantages of foreign protein post-translation processing which is not found in the prokaryotic expression system, and meanwhile avoiding the disadvantages of unstable expression strains, easy to lose plasmid, hyperglycosylation etc. present in the other yeasts. It is a relatively advanced VLP vaccine expression system better than Escherichia coli and the other eukaryotic expression systems.